In the field of biological assays, methods are increasingly sought which allow the limits of detection and analysis of biological entities in biotic fluids to be decreased to lower concentrations, to obtain very high detection sensitivity. Thus, technological improvements have comprised not only the instrumental environment, for example the limits for detecting a signal, but also the design of the sensor reagent itself. Improvements at the signal level have reached a threshold beyond which it is no longer possible to detect the biomolecules, such threshold being of the order of nanomolar (nM) or picomolar (pM).
Nonetheless, other improvements have enabled the signal, undetectable by former technologies, to be measured owing to an amplification of the signal on which the detection principle relies. Such amplification finds its preferred application in the field of biological sensors given that the conditions implemented in biological analysis are compatible with bio-amplification systems.
Currently, two bio-amplification modes are employed in systems intended to detect, for example, immunological reactions. According to a first amplification mode, in ELISA (Enzyme Linked Immunosorbent Assays) tests, the molecule to be detected, for example an antibody that interacts with a chemical entity such as an immobilized antigen is chemically linked to an enzyme. The enzyme is used to catalyze the transformation of the detectable molecules. In the currently used ELISA systems, the enzymes which catalyze the production of chemical entities are almost always hydrolases. The water soluble reaction products are preferably detected in the whole reaction medium by measuring a characteristic property such as absorption, luminescence or bioluminescence.
In biosensors, a second amplification mode is obtained by increasing the number or mass of species detected. This amplification principle is achieved for example by linking mass markers to the molecule to be detected.
If the detection principle relies on fluorescence or absorption, fluorescent or absorbent molecules are chemically linked to the target chemical entity. By way of an example of this type of amplification, U.S. Pat. No. 5,175,270 may be cited, which discloses an amplification mechanism involving a dendrimer architecture at the surface of the sensor. The modified molecule link on each target molecule, or the marked secondary reagent link (for example colloids, nanoparticles or fluorophore-labelled secondary antibodies) will produce linear signal amplification. Latex microspheres, semiconductor nanocrystalline compounds or colloidal gold are mass markers currently used in biosensor systems. In commercial amplification systems, secondary antibodies strongly marked by fluorescent molecules contribute to increasing the signal in a linear manner.
Generally, the systems amplify the sensor signals via reactions catalyzed by an enzyme which increases the number of secondary chemical entities in the medium by catalysis (catalytic amplification for global detection). Otherwise, the sensor signals are increased either by adding mass, for mass sensitive detection, or by increasing the number of labeled molecules, which are linked to the unit.
By way of example of linear amplification at the surface of a sensor, fluorescent signal amplification may be cited. In this system the secondary antibodies are conjugated to allow detection of targets in small quantities. These two amplification modes which have been briefly described above have allowed detection sensitivity to be substantially increased, either in solution or on a surface. They do not, however, allow a sufficiently high signal to be obtained to make them useful in practice.
Binding-pair (also known as ligand-receptor, molecular recognition binding and the like) techniques play an important role in many applications of biomedical analyses and are gaining importance in the fields of environmental science, veterinary medicine, pharmaceutical research, food and water quality control and the like. For the detection of analytes at low concentrations (less than about 1 picomole analyte/sample volume analyzed) the use of fluorescent, luminescent, chemiluminescent, or electrochemiluminescent labels and detection methods are often used.
For the detection of low concentrations of analytes in the field of diagnostics, the methods of chemiluminescence and electrochemiluminescence are gaining widespread use. These methods of chemiluminescence and electrochemiluminescence provide a means to detect low concentrations of analytes by further amplifying the chemical signal of the luminescent molecules many-fold, the resulting “signal amplification” then allowing for detection of low concentrations of analytes.
In addition, the method of Polymerase Chain Reaction (PCR) and other related techniques, have gained wide use for amplifying the number of nucleic acid analytes in the sample. By the addition of appropriate enzymes, reagents, and temperature cycling methods, the number of nucleic acid analyte molecules is amplified such that the analyte can be detected by most known detection means. The high level of commercial activity in the development of new signal generation and detection systems, and the development of new types of test kits and instruments utilizing signal and analyte molecule amplification attests to the importance and need for detection methods with improved sensitivity.